1. Field of the Invention
The present invention relates to WK-5344A substance and WK-5344B substance and production thereof. More particularly, the present invention pertains novel substances, WK-5344A substance and WK-5344B substance having inhibitory action for cholesteryl ester transfer protein and production thereof.
2. Description of Related Art
Various preventive medicaments for myocardial infarction and cerebral accident due to accumulation of cholesterol in vascular wall such as arteriosclerosis caused by adult hypertension have been known. These include, for example, pravastatin, mevinolin and fluvastatin (Endo, A. Journal of Medicinal Chemistry 28, 401-405, 1985 and Endo, A. Journal of Lipid Research 33, 1569-1582, 1992).
Onset of myocardial infarction and cerebral accident and the process of these diseases are very complicate and complex. Consequently, substances having different mechanism of action for treatment of these diseases have been strongly required.
Recently, as a result of westernization of the daily life for foodstuffs, cause of death due to myocardial infarction and cerebral accident as a result of accumulation of cholesterol in the vascular wall is increased and is problem of the life-style related diseases. Most cholesterol is mainly esterified in liver and intestine by long chain fatty acids to form cholesteryl esters, which are secreted into blood as components of chylomicron and very low density lipoprotein, and are circulated in blood mainly as components of low density lipoprotein and high density lipoprotein.
Low density lipoprotein, which transfers cholesterol supplied from the liver into the peripheral tissues, is a risk factor for stimulating arteriosclerosis. On the contrary, the high density lipoprotein collects the cholesterol from the peripheral tissues and is thought to be a factor for suppressing progress of arteriosclerosis. It is cholesteryl ester transfer protein to be involved in the exchange reaction of cholesteryl ester between both lipoproteins, and that is involved in maturation of low density lipoprotein.
Consequently, a substance, which inhibits function of the cholesteryl ester transfer protein, develops decrease of low density lipoprotein, the risk factor for arteriosclerosis, in the blood, and, contrary to that, develops anti-arteriosclerotic action as a result of increase in the high density lipoprotein, which suppresses arteriosclerosis, and is effective for these diseases.
We have studied metabolic product of microorganisms isolated from soils in order to find out novel bioactive substances, and found that cultured mass of newly isolated microbial strain WK-5344 from soil produced substances having inhibitory action on cholesteryl ester transfer protein. As a result of isolation and purification of said cholesteryl ester transfer protein inhibitor from said cultured mass, since such substances having such the chemical structures have not been known, we have designated these substances as WK-5344A substance and WK-5344B substance.
The present invention has been completed accordingly to such knowledge. 
Further object of the present invention is to provide a process for production of WK-5344A substance and WK-5344B substance or salt thereof comprising culturing a microorganism belonging to genus Streptomyces and having ability to produce WK-5344A substance and WK-5344B substance, accumulating WK-5344A substance and WK-5344B substance in a cultured mass and isolating WK-5344A substance and WK-5344B substance from said cultured mass.
According to the preferred embodiment of the present invention, the present invention pertains the process for production of novel substance, WK-5344A substance and WK-5344B substance, or salt thereof wherein the microorganism belonging to genus Streptomyces having ability to produce WK-5344A substance and WK-5344B substance is Streptomyces sp. WK-5344. The present invention further relates to a microorganism belonging to genus Streptomyces having ability to produce WK-5344A substance and WK-5344B substance and said microorganism is Streptomyces sp. WK-5344 (FERM BP-6668).
The microorganism having ability to produce WK-5344A substance and WK-5344B substance of the present invention (hereinafter designates as WK-5344 substance producing microorganism) belongs to genus Streptomyces, and is sufficient to have ability to produce WK-5344A substance and WK-5344B substance without no limitation. Example of the preferable microbial strain used to produce WK-5344A substance and WK-5344B substance of the present invention is Streptomyces sp. WK-5344, which is isolated from newly collected soil by the present inventors.
The taxonomical properties of the present strain are as follows.
(I) Morphological Properties
Vegetative mycelia grow well in various agar media and no fragmentation was observed. Aerial mycelia grow abundantly on inorganic salts-starch agar and glycerol-asparagine agar to show white to gray color. In microscopic observation, aerial mycelia showing spiral and chains of more than 20 spores are observed. Form of spores is oval with size of 1.0xc3x970.5 xcexcm. Surface of spore is spiny. No sclerotium, sporangium and zoospore are observed.
(II) Cultural Properties on Various Agar Media
The cultural properties of the present producing strain observed according to the method of E. B. Shirling and D. Gottlieb (International Journal of Systematic Bacteriology, 16, 313, 1966) are shown in Table 1.
Color tones are determined referring to Color Harmony Manual, 4th ED. (Container Corporation of America, Chicago, 1958) as standard color, and are indicated as name of color tone with its cord in parenthesis. The following table indicates, if not specifically defined, results of visual observation of the state of culture of this strain in various culture media at 27xc2x0 C. for 2 weeks.
(IV) Composition of Cell Wall
Diaminopimelic acid of cell wall is LL type.
Taxonomical properties of the present strain are summarized as follows.
Diaminopimelic acid in cell wall is LL type. Vegetative mycelia show good growth on various agar media and no fragmentation was observed. Form of aerial mycelia is spiral with long spore chain. Surface of spore is spiny. Various properties on culture are showing brownish color tone of vegetative mycelia and white to gray color tone of aerial mycelia. Soluble pigment formation is greenish yellow pigment on yeast extract-malt extract agar medium, oatmeal agar medium, glucose-asparagine agar medium, glycerol-asparagine agar medium, glucose-nitrate agar medium and glycerol-calcium malate agar medium.
According to results of these observations, the present strain has been identified as a strain belonging to genus Streptomyces and was referred to Streptomyces sp. WK-5344.
The present strain was deposited in the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, located at AIST Tsukuba Central 6, 1-1, Higashi 1-chome Tsukuba-shi, Ibaraki-ken, 305-8566 Japan, as FERM BP-6668 on Mar. 1, 1999.
Although WK-5344 substance producing microbial strain was explained as a preferable strain of the present invention, taxonomical properties are very easily mutated as general properties of microorganisms and are not stable. It is also well known to mutate by natural mutation technique or commonly performed artificial mutation techniques such as ultraviolet irradiation, X-ray irradiation or applying mutagenic agents such as N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine and ethyl methanesulfonate. Consequently, strains having ability to produce WK-5344 substance belonging to genus Streptomyces including artificial mutants and natural mutants can be used in the present invention. Strains mutated by cell engineering technology such as cell fusion and gene manipulation are included within the WK-5344 substance producing strain.
In production of WK-5344A substance and WK-5344B substance of the present invention, WK-5344 substance producing microorganism belonging to genus Streptomyces is cultured in preferable medium. In the culture of the present microorganism, culture method for common microorganism is generally provided. Examples of media are those nutrient media containing assimilable carbon sources for microorganism, digestible nitrogen sources for microorganism and, if necessary, adding inorganic salts.
Examples of the above assimilable carbon sources are glucose, sucrose, molasses, starch, dextrin, cellulose, glycerol and organic acid, and are used in alone or in combination. Examples of digestible nitrogen sources are organic nitrogen source such as peptone, meat extract, yeast extract, dry yeast, soy bean powder, corn steep liquor, cotton seed oil, casein, soy bean protein hydrolysate, amino acids and urea, and inorganic nitrogen source such as nitrate and ammonium salt, and are used in alone or in combination.
Further, if necessary, inorganic salt such as sodium salt, potassium salt, calcium salt, magnesium salt and phosphate, and heavy metal salt are added. In the medium, trace nitrogen element, growth stimulating substance or precursor for stimulating growth of the present strain and production of WK-5344A substance and WK-5344B substance can be added.
Cultivation can be performed under aerobic condition such as shaking culture or aeration stirring culture. pH of the medium is preferably performed at neutral condition. For idustrial production, submerged aeration is preferable. Culturing condition can be performed within range of 20-37xc2x0 C., generally at 24-30xc2x0 C. preferably at 27xc2x0 C. Culturing time is generally for 2-6 days in the liquid culture to accumulate WK-5344A substance and WK-5344B substance, and the cultivation can be terminated when maximum production of WK-5344A substance and WK-5344B substance can be performed.
The culture condition such as composition of medium, culturing temperature, rate of stirring and aeration volume can be adjusted and selected in order to obtain preferable conditions depending on types of strain used and external conditions. In the liquid culture, if foaming occurs, antifoaming agents such as silicon oil, vegetable oil and surface active agents can be used.
Since WK-5344A substance and WK-5344B substance accumulated in the cultured mass are contained in the cultured liquid or cultured mycelia, the cultured mass is filtered by adding filter aid such as Celite and Hyflosupercell or centrifuged to separate culture liquid and mycelia, which are extracted with organic solvent, then the extracts are advantageously concentrated and isolated WK-5344A substance and WK-5344B substance.
Isolation of WK-5344A substance and WK-5344B substance from the culture filtrate is performed by extracting the culture filtrate with water immiscible organic solvent such as ethyl acetate, butyl acetate and benzene and concentrating the extract in vacuo to obtain WK-5344A substance and WK-5344B substance. Said crude substance can be purified by known method used for purification of lipophilic substance, for example column chromatography using carrier such as silica gel or alumina to obtain purified WK-5344A substance and WK-5344B substance.
In order to isolate WK-5344A substance and WK-5344B substance from mycelia, the mycelia are extracted with aqueous water miscible organic solvent such as aqueous methanol, concentrated the extract in vacuo, extracted the concentrate with water immiscible organic solvent such as ethyl acetate, butyl acetate or benzene, and purified the extract with or without combining the above extract obtained from the culture liquid to obtain WK-5344A substance and WK-5344B substance.
Physico-chemical properties of WK-5344A substance and WK-5344B substance of the present invention are described as follows.
[I] WK-5344A Substance
(1) Molecular formula: C45 H30N3 O13Fe [in high resolution FAB mass spectrum (positive), m/z 877.1203 (M+H) is measured] (calculated: 877.1206)
(2) Molecular weight: 876 [m/z 877 (M+H)+and 899 (N+Na)+are observed by FAB mass spectrum (positive)]
(3) Specific rotation: [xcex1]D 25 xe2x88x923000xc2x0 (c=0.01, methanol)
(4) UV spectrum: UV spectrum measured in methanol is shown in FIG. 1. Specific maximum peaks are observed at around 280, 305 (shoulder), 440 and 690 nm.
(5) IR spectrum: IR spectrum (KBr tablet) is shown in FIG. 2. xcexmax KBr cmxe2x88x921: 3400, 1728, 1701, 1597, 1493, 1385, 1284, 1207 and 1105.
(6) Solubility in solvent: Soluble in methanol, ethanol, acetonitrile, ethyl acetate, chloroform and dimethyl sulfoxide. Insoluble in water.
(7) Nature of substance: neutral
(8) Color and form substance: greenish powder
(9) Proton NMR: FIG. 3 (Varian NMR, in deuterium methanol, 400 MHz)
(10) Carbon NMR: FIG. 4 (Varian NMR in deuterium methanol, 100 MHz)
Structure of WK-5344A substance was determined by considering the above physico-chemical properties and spectral data as follows. 
[II] WK-5344B Substance
(1) Molecular formula: C46 H30N3 O14Fe [in high resolution FAB mass spectrum (positive), m/z 905.1151 (M+H) is measured] (calculated: 905.1155)
(2) Molecular weight: 904 [m/z 905 (M+H)+ and observed by FAB mass spectrum (positive)]
(3) Specific rotation: [xcex1] D 25 xe2x88x921000xc2x0 (c=0.01, methanol)
(4) UV spectrum: UV spectrum measured in methanol is shown in FIG. 5. Specific maximum peaks are observed at around 285, 303 (shoulder), 445 and 698 nm.
(5) IR spectrum: IR spectrum (KBr tablet) is shown in FIG. 6. xcexmax KBr cmxe2x88x921: 3400, 1718, 1701, 1597, 1508, 1385, 1281, 1196 and 1105.
(6) Solubility in solvent: Soluble in methanol, ethanol, acetonitrile, ethyl acetate, chloroform and dimethyl sulfoxide. Insoluble in water
(7) Nature of substance: neutral
(8) Color and form of substance: greenish powder
(9) Proton NMR: FIG. 7 (Varian NMR, in dueterium methanol, 400 MHz)
(10) Carbon NMR: FIG. 8 (Varian NMR, in dueterium methanol, 100 MHz)
Structure of WK-5344B substance was determined by considering the above physico-chemical properties and spectral data as follows. 
As described hereinabove, physico-chemical properties of WK-5344A substance and WK-5344B substance were explained in details, no compounds identical with these properties have not been reported, and WK-5344A substance and WK-5344B substance are determined as novel substances.
Biological properties and inhibitory activity of WK-5344A substance and WK-5344B substance of the present invention are explained hereinbelow.
(1) Inhibitory Action for Human Cholestryl Ester Transfer Protein
Effect on cholesteryl ester transfer protein is determined using crude protein prepared from human plasma according to a method described in Kato, et al. Journal of Biological Chemistry, 264, 4082-4087, 1989.
Reconstructed high density lipoprotein (hereinafter designates as HDL) containing [1xe2x88x9214C] cholesteryl ester 25 xcexcl, human originated low density lipoprotein (hereinafter designates as LDL) 10 xcexcl, 7 mM 5,5-dithiobisnitrobenzoic acid 30 xcexcl, and partially purified human cholesteryl ester transfer protein 5 xcexcl were combined. Total 150 xcexcl reaction mixture was reacted at 37 xc2x0 C. for 30 minutes.
After the reaction, 0.1% dextran sulfate 5 xcexcl, 6 mM MgCl2 5 xcexcl and 20 xcexcl of phosphate buffer adjusted to ionic strength 0.16 were added therein and the mixture was allowed on ice for 20 minutes. The mixture was centrifuged at 4xc2x0 C., 13000 rpm for 15 minutes. The precipitated LDL fraction was collected. LDL was dissolved in 0.1 N NaOH 180 xcexcl and the transferred cholestryl ester in LDL was measured by liquid scintillation counter. Results of 50% inhibition for cholesteryl ester transfer protein activity were 0.54 xcexcg/ml of WK-5344A substance and 2.0 xcexcg/ml of WK-5344B substance.
As described hereinabove, WK-5344A substance and WK-5344B substance of the present invention show significant inhibitory action against cholesteryl ester transfer protein and are thought to be useful for prevention and treatment of diseases caused by accumulation of cholesterol in human.